RNA interference (or “RNAi”) is currently recognized as a highly specific mechanism of sequence-specific gene silencing. See deFougerolles et al. (2007) Nature Reviews 6:443-453. The mechanism allows for the specific and profound reduction of proteins and mRNA.
Briefly, the RNAi mechanism involves double-stranded RNA (dsRNA) intentionally synthesized with a sequence complementary to a gene of interest and subsequently introduced into a cell or organism, where the dsRNA is recognized as exogenous genetic material and activates the RNAi pathway. If the exogenous dsRNA is relatively long, it will be cleaved into small interfering RNAs (siRNAs). Alternatively, if the exogenous dsRNA is relatively short (about 30 base pairs or less), cleavage does not occur, the exogenous dsRNA itself acts as the siRNA substrate, and complications arising from activation of innate immunity defenses are avoided. In both cases, the siRNA becomes incorporated into an RNA-induced silencing complex (RISC) followed by unwinding of the double stranded siRNA into two strands. One of these strands, the “sense” strand (also known as the “passenger” strand), is discarded. The other strand, the “guide” strand (also known as the “antisense” strand) recognizes target sites to direct mRNA cleavage, thereby silencing its message. A similar RNAi mechanism involves microRNAs (miRNAs) deriving from imperfectly paired non-coding hairpin RNA structures.
Through the specific targeting of genes, RNAi-based therapies have the ability to substantially block the production of undesired proteins. Thus, in diseases and conditions attributable to the undesired or over expression of certain proteins, RNAi-based therapies represent a potentially powerful and important approach in medical therapies.
Despite the great promise of RNAi-based therapies, there remains a problem of the relative short half life of the small interfering nucleic acids “siNAs” used in RNAi-based approaches in vivo. Thus, there remains a need for better and improved versions of siNAs in order to bring the RNAi-based therapies to fruition.
Chitosan-PEG conjugates have been used as complexing agents for siRNA. See, for example, WO10/021,720 and WO10/021,718. In the studies described therein, PEG-chitosan conjugates were prepared by reaction of a PEG with an amine group on the chitosan. Because a chitosan molecule includes numerous amine groups, PEG molecules conjugated to these amine groups produce mixtures of PEGylated chitosans, each having a different number of PEG molecules conjugated to the chitosan and each generating different complexing properties. As a consequence, it is not possible to prepare a well defined product based on conjugating PEG to the numerous amine groups of chitosan. It would therefore be desirable to form a PEG-chitosan conjugate in which a single PEG molecule could be attached to the carbohydrate. Among other things, such a PEG-chitosan conjugate could be formed in a more uniform and consistent way, thereby having the advantage of (among other things) more uniform and consistent siNA compositions, including: (a) complexes of siNA and monoPEG-chitosan; and (b) conjugates of siNA wherein siNA is conjugated to monoPEG-chitosan. More uniform and consistent siNA compositions will have the advantages of more uniform and consistent performance, and (at least with respect to complexes) more stable complex formation.